富含n-3多不饱和脂肪酸的肠外营养对脓毒症大鼠免疫功能影响及机制研究

The effects of parenteral nutrition enriched with n-3 multi-unsaturated fatty acids (PUFAs) on the immune function during sepsis and its possible mechanism remain unclear. In this study, rats are subjected to cecal ligation and puncture-induced sepsis. Rats are randomly assigned to receive normal saline, or total parenteral nutrition containing a standard soybean oil emulsion, or fish oil-supplemented total parenteral nutrition at the onset of sepsis for 5 days. The survival rate and body weight change are investigated at day 5 after CLP or sham operation. Moreover, to determine the effects of parenteral FO on the local and systemic bacterial loads, bacterial culture from peritoneal lavage fluid, blood, lung, and spleen are performed in different groups. Flow cytometry is used to determine the proportions of the blood and spleen lymphocyte subsets. The comparisons in CD3+, CD3+CD4+, CD3+CD8+ and CD4+CD25+Foxp3+ T lymphocytes subsets, as well as the ratio of CD3+CD4+ to CD3+CD8+ T cells, are analyzed. Foxp3 mRNA and protein levels in all groups are determined by RT-PCR and Western-blot. The plasma HMGB1 and IL10 levels are tested by ELISA. Additionally, gas chromatography is used to analyze the fatty acids profile in plasma and erythrocyte membranes. TLR4, MYD88 and NF-κB mRNA and protein levels are determined by RT-PCR and Western-blot.

富含n-3多不饱和脂肪酸的肠外营养对脓毒症免疫功能的影响及其可能的作用机制仍不明确。本研究通过盲肠结扎穿孔法制作大鼠脓毒症模型，通过颈静脉置管持续给予富含n-3多不饱和脂肪酸（n-3 PUFAs）的肠外营养治疗5天，观察n-3PUFAs对脓毒症大鼠生存率和体重影响；观察肺脏、肝脏、脾脏的病理改变，以及局部和全身细菌负荷变化，评估不同作用因素的抗菌能力；通过检测血和脾脏主要T淋巴细胞亚群分布、CD4+CD25+Foxp3+调节性T细胞(Regulatory T Cells, Treg)比例与功能，ELISA法检测HMGB1和IL10的浓度，探讨n-3PUFAs对脓毒症免疫功能可能的调理作用；气相色谱法检测血浆和红细胞膜主要磷脂脂肪酸组成改变，检测单个核细胞TLR4、MYD88、NF-κB(p65) mRNA和蛋白表达变化，揭示n-3PUFAs对脓毒症免疫功能调理的可能作用机制。

脓毒症是ICU最常见的死因，其死亡率高，预后差。尽管随着国际指南的提出及修改，脓毒症患者的预后有所改善，然而其死亡率仍然居高不下，预后不理想。我们通过建立脓毒症大鼠模型，对4组（对照组、生理盐水组、鱼油组及大豆油组）大鼠外周血淋巴亚群、急性肺损伤、腹腔冲洗液细菌负荷及脾脏Foxp3的表达等指标进行分析。研究结果提示，富含n-3 PUFAs的肠外营养治疗提高了脓毒症大鼠的存活率，减轻了重要脏器的病理损害和降低了细菌负荷，脓毒症导致血和脾脏T淋巴细胞亚群比例失调，导致免疫功能紊乱；脓毒症早期给予富含n-3PUFAs的肠外营养治疗可发挥免疫保护作用，并可调理炎症介质的平衡。FO组肺干重湿重比例明显升高；小肠粘膜病理损伤评分明显降低；脾脏TLR-4表达明显降低，MYD88及NF-κB（p65）表达无明显差异，但磷酸化NF-κB（p65）表达有所下降；小肠组织occludin表达明显降低；血清HMGB-1和TNF-α浓度有所下降。本课题验证了富含n-3多不饱和脂肪酸的肠外营养能够调节脓毒症大鼠免疫功能，降低炎症反应及急性肺损伤程度，并对其机制进行了探讨，为临床脓毒症治疗提供新的视角及理论依据。